Pharmaceutical formulations of tropomyosin related kinase (TRK) inhibitors

ABSTRACT

Pharmaceutical formulations with a tropomyosin-related kinase inhibitor (“Trk inhibitor”) are disclosed. The pharmaceutical formulations comprise 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine in microcrystalline suspension formulations in its monohydrate form, which shows improved characteristics over the anhydrate form, and in extended release formulations. The extended release pharmaceutical formulations comprise 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loaded microspheres.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 15/108,686,filed Jun. 28, 2016 which is a 371 National Phase Entry application ofInternational Application No. PCT/US2014/069338, filed Dec. 9, 2014,which claims the benefit under 35 U.S.C. § 119 of U.S. ProvisionalApplication 61/914,842, filed Dec. 11, 2013, all of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to pharmaceutical formulations of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,a tropomyosin-related kinase inhibitor (“Trk inhibitor”), and amonohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminemonohydrate. The monohydrate form has desirable properties whichfacilitate the preparation of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineinto pharmaceutical formulations.

The Trk inhibitor microcrystalline solution pharmaceutical formulationscomprise3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein its monohydrate form, which shows improved characteristics over theanhydrate form.

The instant invention also relates to extended release pharmaceuticalformulations of the Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,comprising3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrospheres.

This invention further relates to the use of these pharmaceuticalformulations to treat diseases including inflammatory diseases,autoimmune disease, defects of bone metabolism, and cancer, as well asin the treatment of osteoarthritis (OA), pain, post-operative pain, andpain associated with OA.

The Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand methods of producing the Trk inhibitor are disclosed inInternational Patent Application Number PCT/US14/69469 and U.S. patentapplication Ser. No. 14/564,773, each entitled Tropomyosin RelatedKinase (TRK) Inhibitors, which are incorporated herein by reference.

Related Art

Not applicable

BRIEF SUMMARY OF THE INVENTION

A first aspect of the invention relates to a crystalline form of the Trkinhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,wherein the x-ray powder diffraction pattern contains the following 2θpeaks measured using CuK_(α) radiation: 7.14, 8.89, 10.22, 12.42, 12.73and 14.31.

A second aspect of the invention relates to pharmaceutical formulationscomprising the crystalline form of the Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,wherein the x-ray powder diffraction pattern contains the following 2θpeaks measured using CuK_(α) radiation: 7.14, 8.89, 10.22, 12.42, 12.73and 14.31, and a pharmaceutically acceptable excipient.

A third aspect of the invention relates to the monohydrate form of theTrk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.

A fourth aspect of the invention relates to pharmaceutical formulationscomprising the monohydrate form of the Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand a pharmaceutically acceptable excipient.

A fifth aspect of the invention relates to extended releasepharmaceutical formulations comprising3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrospheres.

In a sixth aspect, the invention relates to methods of manufacturing acrystalline form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminecomprising:

-   -   a. Mixing        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        with a solvent to form a suspension;    -   b. Stirring the suspension;    -   c. Collecting the solids in the suspension by filtration; and    -   d. Drying the solids.

In a seventh aspect, the invention relates to methods of manufacturing amonohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminecomprising:

-   -   a. Mixing        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        with a solvent to form a suspension;    -   b. Stirring the suspension;    -   c. Collecting the solids in the suspension by filtration; and    -   d. Drying the solids.

In an eighth aspect, the invention relates to methods of manufacturing3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrocapsules by solvent extraction, comprising:

-   -   a. Dissolving the        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        in an organic solvent to form a drug solution;    -   b. Adding a polymer to the drug solution to form a        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        solution;    -   c. Mixing the        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        solution into an aqueous solution to form an emulsion;    -   d. Adding deionized water to the emulsion;    -   e. Forming microspheres from the emulsion by solvent extraction;        and    -   f. Sieving the resulting microspheres using a surfactant        solution.

And in a ninth aspect, the invention relates to methods of manufacturing3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrocapsules by solvent extraction, comprising:

-   -   a. Dispersing the        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        in an organic solvent to form a drug suspension;    -   b. Adding a polymer to the drug suspension to form a        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        dispersion;    -   c. Mixing the        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        dispersion with an aqueous solution to form an emulsion;    -   d. Adding deionized water to the emulsion;    -   e. Forming microspheres from the emulsion by solvent extraction;        and    -   f. Sieving the resulting microspheres using a surfactant        solution.

In an eleventh aspect, the invention relates to methods of manufacturing3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrocapsules by spray drying, comprising:

-   -   a. Dissolving the        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        in an organic solvent to form a drug solution;    -   b. Adding a polymer to the drug solution to form a        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        solution; and    -   c. Pumping the        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        solution through a sprayer into a dryer to form a spherical        particle.

In a twelfth aspect, the invention relates to methods of manufacturing3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrocapsules by spray drying, comprising:

-   -   a. Dispersing the        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        in an organic solvent to form a drug suspension;    -   b. Adding a polymer to the drug suspension to form a        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        dispersion; and    -   c. Pumping the        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        dispersion through a sprayer into a dryer to form a spherical        particle.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1: X-Ray Powder Diffraction (XRPD) of the Monohydrate Form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,4-40° 20. The XRPD of the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineshows unique peaks at 7.14, 8.89, 10.22, 12.42, 12.73 and 14.31 2θ peaksmeasured using CuK_(α).

FIG. 2: Differential Scanning calorimetry (DSC) and Thermal GravimetricAnalysis (TGA) Thermogram of the Monohydrate Form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,Heated at 10° C./min. The DSC thermogram exhibits three thermal eventsat 76.72, 160.13, and 195.78° C., the TGA thermogram shows 3.7% weightloss from 25-100° C.

FIG. 3: Crystalline Structure of the Monohydrate Form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.The crystalline structure of the monohydrate form of the Trk inhibitoris displayed.

FIG. 4: Overlay of Experimental XRPD Pattern with the XRPD PatternCalculated from the Single Crystal Structure. This figure displays anoverlay of the experimental powder pattern with the one calculated fromthe single crystal structure. The strong degree of matching suggeststhat the single crystal structure is indicative of the bulk material.

FIG. 5: Effect of API Loading on IVR Profile—12% API/9:1R202H:752H, 16%API/9:1 R202H:752H and 20% API/9:1 R202H:752H Microspheres. Effect ofAPI Loading on IVR Profile—12% API/9:1 R202H:752H, 16% API/9:1R202H:752H and 20% API/9:1 R202H:752H Microspheres. This figure comparesthe effect of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineloading on the in vitro release profile of microspheres of varyingcomposition.

FIG. 6: DSC Thermograms of 16% API/9:1 R202H:752H and 20% API/9:1R202H:752H Microspheres. The DSC thermogram demonstrates that 20%microspheres show a melting endotherm between 130-150° C. confirming thepresence of surface drug crystals.

FIG. 7: Scanning Electron Microscopy (SEM) View of 16% API/9:1R202H:752H Microspheres (1500×). Scanning Electron Microscopy (SEM) Viewof The 16% drug-loaded microspheres show no drug crystals, indicatingthat the drug is amorphous.

FIG. 8: Scanning Electron Microscopy (SEM) View of 20% API/9:1R202H:752H Microspheres (1500×). The 20% drug-loaded microspheres showsurface drug crystals.

FIG. 9: Effect of API Loading on IVR Profile—15% API/R202H, 17%API/R202H and 19% API/R202H Microspheres. This figure compares theeffect of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineloading on the in vitro release profile of microspheres of varyingcomposition.

FIG. 10: Effect of API Loading on IVR Profile—16% API/9.5:0.5R202H:752H, 18% API/9.5:0.5 R202H:752H and 20% API/9.5:0.5 R202H:752HMicrospheres. This figure compares the effect of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineloading on the in vitro release profile of microspheres of varyingcomposition.

FIG. 11: Effect Polymer Blend on IVR Profile—16% API/R202H, 16%API/9.0:0.5 R202H:RG752H and 16% API/9:1 R202H:RG752H Microspheres. Thisfigure compares the effect of the polymer blend on the in vitro releaseprofile of microspheres of varying composition.

FIG. 12: Effect Polymer Blend on IVR Profile—16% API/R202H, 16%API/9.5:0.5 R202H:RG502H and 16% API/9:1 R202H:RG502H Microspheres. Thisfigure compares the effect of the polymer blend on the in vitro releaseprofile of microspheres of varying composition.

FIG. 13: Formulations Showing Zero-Order IVR Profile for 180 Days—15%API/9:1 R203H:RG752H and 16% API/9.5:0.5 R202H:RG502H Microspheres.Microspheres prepared using 15%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R203H:752H polymers at a ratio of 9:1 and 16%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202H:RG502H polymers at a ratio of 9.5:0.5 exhibited a pseudozero-order release profile over 6 months.

FIG. 14: Effect of Co-Solvent Systems in Preparation on IVR Profile—16%API/9:1 R203H:RG752H Microspheres—9:1 DCM:MeOH, 9:0.5:0.5 DCM:MeOH:BA,9.5:0.05 DCM:BA, 9:1 DCM:BA. This figure compares the effect of theco-solvent system used in the preparation of the microspheres on the invitro release profile of microspheres with 16% drug-loading and apolymer blend of 9:1 R203H:RG752H.

FIG. 15: Effect of DCM:BA Co-Solvent System to Increase API Loading onIVR Profile—16% API/9:1 R202H:RG752H, 25% API/9:1 R202H:RG752H, 30%API/R202H, 40% API/R202H, 25% API/R203H, 30% API/R203H, 40% API/R203H,50% API/R203H Microspheres. This figure compares the effect of theco-solvent system used in the preparation of the microspheres toincrease drug-loading on the in vitro release profile of microspheresvarying polymer blends.

FIG. 16: Scanning Electron Microscopy (SEM) View of MicrospheresPrepared with Micronized Suspension Microencapsulation Process (1500×).Microspheres produced by encapsulating a suspension of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewere spherical with a rough surface texture due to the presence of drugcrystals embedded in the surface.

FIG. 17: Effect of Micronized Suspension Microencapsulation Process onIVR Profile—25% API/9:1 R202H:RG752H 39 mL 5% PVA+2.6 mL EA, 25% API/9:1R202H:RG752H 39 mL 5% PVA+3.25 mL EA, 25% API/R203H 39 mL 5% PVA+2.0 mLEA, 30% Microfluidized API/9:1 R202H:RG752H 39 mL 5% PVA+2.0 mL EA, 40%Microfluidized API/9:1 R202H:RG752H 39 mL 5% PVA+2.0 mL EA, 30%Microfluidized API/9:1 R202H:RG752H 39 mL 5% PVA+2.0 mL EA, 30%Microfluidized API/9:1 R202H:RG752H 39 mL 5% PVA+2.3 mL EA, 30%Microfluidized API/R202S 39 mL 5% PVA+2.0 mL EA, 30% MicrofluidizedAPI/R203S 39 mL 5% PVA+2.0 mL Microspheres. This figure compares themicronized suspension microencapsulation process used in the preparationof the microspheres on the in vitro release profile of microspheres withvarying drug-loading.

FIG. 18: Scanning Electron Microscopy (SEM) View of 16% API/9:1R202H:RG752H Microspheres, Solvent Extraction (1000×). Solventextraction microspheres were spherical with a smooth surface texture.

FIG. 19: Scanning Electron Microscopy (SEM) View of 16% API/9:1R202H:RG752H Microspheres, Spray Drying (1000×). Spray driedmicrospheres were spherical with some surface texture.

FIG. 20: Effect of Microencapsulation Process on IVR Profile—16% API/9:1R202H:RG752H Microspheres by Solvent Extraction (OW), 16% API/9:1R202H:RG752H Microspheres by Spray Drying (20% and 22.5% Polymer) and16% API/9:1 R203H:RG752H Microspheres by Spray Drying (22.5% Polymer).This figure compares the in vitro release profile of 16% drug-loaded,9:1 R202H:RG752H microspheres prepared by solvent extraction and spraydrying.

FIG. 21: Scanning Electron Microscopy (SEM) View of 16% API/1:1R202H:R203H/No Additive Microspheres, Spray Dried (1000×). This figureshows the effect of the microencapsulation process on the on the invitro release profile of microspheres of varying formulations.

FIG. 22: Scanning Electron Microscopy (SEM) View of 16% API/1:1R202H:R203H/31.25 mg PEG Microspheres, Spray Dried (1000×). Spray driedmicrospheres of 16% API/1:1 R202H:R203H/No Additive are presented.

FIG. 23: Scanning Electron Microscopy (SEM) View of 16% API/1:1R202H:R203H/31.25 mg Poloxamer 407 Microspheres, Spray Dried (1000×).Spray dried microspheres of 16% API/1:1 R202H:R203H/31.25 mg PEG arepresented.

FIG. 24: Effect of % 10 kDa PEG or 1% Poloxamer 407 on IVR Profile—16%API/1:1 R202H:R203H/No Additive, 16% API/1:1 R202H:R203H/31.25 mg PEG,16% API/1:1 R202H:R203H/31.25 mg Poloxamer 407. Spray dried microspheresof 16% API/1:1 R202H:R203H/31.25 mg Poloxamer 407 are presented.

FIG. 25: In vivo (Rat) IVR Profile—16% API/9:1 202H:RG502H and 15%API/9:1 R203H:RG752H Microspheres. This figure compares the in vitrorelease profile of 16% API/1:1 R202H:R203H/No Additive microspheres, 16%API/1:1 R202H:R203H/31.25 mg PEG microspheres, and 16% API/1:1R202H:R203H/31.25 mg Poloxamer 407 prepared by spray drying.

FIG. 26: [¹⁴C]3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineRemaining Over Time (Rat Knee Joint)—16% API/9:1 202H:RG502H and 15%API/9:1 203H:RG752H Microspheres. This figure shows a near-zero-orderrelease in vivo over approximately 3-4 months and 5-6 months for 16%API/9:1 202:H:RG502H and 15% API/9:1 R203H:RG752H microspheres.

FIG. 27:3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineConcentration-Time Profiles (Rat Blood)—16% API/9:1 202:H:RG502H and 15%API/9:1 R203H:RG752H Microspheres. Following intra-articularadministration into rat knee joints, for 16% API/9:1 202:H:RG502H and15% API/9:1 R203H:RG752H microspheres showed drug release over 5 to 6months; 16% API/9:1 202:H:RG502H showed 12% remaining in the joint after5 months and 15% API/9:1 R203H:RG752H showed 30% of the drug remainingafter 6 months.

FIG. 28: IVR Profile—16% API/9:1 202:H:RG752H, 15% API/9:1 R203H:RG752Hand 40% API/203H Microspheres. This figure shows the drugconcentration-time profile in blood following intra-articularadministration of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrospheres.

FIG. 29:3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineConcentration-Time Profiles—16% API/9:1 202:H:RG752H, 15% API/9:1R203H:RG752H and 40% API/203H Microspheres.

FIG. 30: Measured or Simulated XRPD of Forms 1 to 4 (Anhydrous andHydrate).

FIG. 31: XRPD of Form 5 (Ethanol) of3-(3-methoxy-4-((4-methoxybenzl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis presented.

FIG. 32: XRPD of Form 9 (Acetone) of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis presented.

FIG. 33: XRPD Comparison of Form 10 (Acetone) and Form 9 (Acetone). Thisfigure provides a comparison of the two forms of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineobtained in acetone.

FIG. 34: XRPD of Form 11 (Acetonitrile). The XRPD of Form 11(Acetonitrile) of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis presented.

FIG. 35: Solid Crystalline Phases of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.This figure provides a comparison of the ten crystalline forms of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineobtained in experiments.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to pharmaceutical formulations of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,a tropomyosin-related kinase inhibitor (“Trk inhibitor”), and amonohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.

The Trk inhibitor microcrystalline solution pharmaceutical formulationscomprise3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein its monohydrate form, which shows improved characteristics over theanhydrate form.

The instant invention also relates to extended release pharmaceuticalformulations of the Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,comprising3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrospheres.

This invention further relates to the use of these pharmaceuticalformulations to treat diseases including inflammatory diseases,autoimmune disease, defects of bone metabolism, and cancer, as well asin the treatment of osteoarthritis (OA), pain, post-operative pain, andpain associated with OA.

The pharmaceutical formulations of the Trk inhibitor of the instantinvention inhibit tropomyosin-related kinase A (TrkA),tropomyosin-related kinase B (TrkB), tropomyosin-related kinase C(TrkC), and c-FMS (the cellular receptor for colony stimulating factor-1(CSF-1)). Tropomyosin-related kinases (Trk) are high affinity receptorsactivated by solubule growth factors called neutrophins (NT). TrkA, alsoknown as neurotrophic tyrosine kinase receptor type 1, is activated bynerve growth factor (NGF). TrkB is activated by brain derived growthfactor and NT-4/5. TrkC is activated by NT3. The activation of Trk leadsto the activation of downstream kinases that are implicated in cellsignaling, including cell proliferation, survival, angiogenesis andmetastasis. Trk have been implicated in a number of diseases, includingOA.

The pharmaceutical formulations of the Trk inhibitor of the instantinvention can also inhibit c-FMS (the cellular receptor for colonystimulating factor-1 (CSF-1). C-FMS plays a role in the regulation ofmacrophage function, and is believed to play a role in inflammatorydiseases, autoimmune disease, defects of bone metabolism and cancer(Burns and Wilks, 2011, Informa Healthcare).

OA is a prevalent and debilitating joint disease characterized bychronic pain and destruction of articular cartilage. Recent clinicaltrials have confirmed a role for blocking NGF in OA knee pain,demonstrating significant pain relief and high responder rates inpatients treated by intravenous infusion with anti-NGF blockingantibodies (Lane, 2010, N Engl J Med). However, this modality may leadto an increased risk for adverse events due to systemic inhibition ofNGF signaling (FDA Arthritis Advisory Committee Meeting to DiscussSafety Issues Related to the Anti-Nerve Growth Factor Agents;http://www.fda.gov/AdvisoryCommittees/Calendar/ucm286556.htm)Accordingly, a novel approach toward targeting NGF-mediated OA pain hasbeen adopted through the development of Trk inhibitors, specificallyTrkA inhibitors, the high-affinity receptor for NGF (Nicol, 2007,Molecular Interv). The Trk inhibitors of the present invention aredelivered locally and thereby avoid the systemic distribution observedwith intravenous anti-NGF administration. This treatment strategyprovides enhanced dosing convenience, as well greater safety by allowingfor the maintenance of physiologically necessary NGF signaling (i.e.sensory/sympathetic nerve maintenance, angiogenesis) at non-local sites.

This invention relates to pharmaceutical formulations of the Trkinhibitor and methods of treating disease with pharmaceuticalformulations of the Trk inhibitor. More particularly, the inventionpertains to methods of treating pain, OA, pain associated with OA,post-operative pain, inflammatory diseases, autoimmune disease, defectsof bone metabolism and cancer with pharmaceutical formulations of theTrk inhibitors. The pharmaceutical compositions of Trk inhibitors can beadministered in multiple dosage forms, including an injection for localdelivery both as a microcrystalline suspension and in extended releaseformulations. The Trk inhibitor is the active pharmaceutical ingredientin pharmaceutical compositions comprising the Trk inhibitor. The Trkinhibitor can also be co-administered and/or co-formulated with otheractive ingredients for the treatment of disease, including the treatmentof pain, OA and pain associated with OA.

The pharmaceutical formulations of the Trk inhibitor of the presentinvention comprise3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,represented by Formula (I) below, as the active pharmaceuticalingredient.3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis also known as GZ389988.

Formula (I):3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine(GZ389988)

The problem to be solved with this invention is the difficulty informulating compositions comprising3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.The solution to this problem is the discovery that the monohydrate formof3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminehas improved physical and chemical properties, including better physicalstability and slower aqueous dissolution, compared to the anyhdrateform. The anhydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineproved relatively unstable in solution, yielding issues with itsformulation into a pharmaceutical composition. The anhydrate formexhibits variable solid form changes under certain conditions, includingconversion to the hydrate. The monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineallowed for additional stability and is essential to avoid polymorphicconversion upon long term storage and during processing, and lead toenhanced physical stability. Further, slower dissolution in aqueoussolution with the monohydrate form was observed. The monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,also known as GZ389988A, is represented by Formula (II) below.

Formula (II): Monohydrate Form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine(GZ389988A)

The molecular weight of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis 470.54 g/mol, and the elemental formula is C₂₆H₂₆N₆O₃. Themonohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis an off-white powder and under a polarized light microscope it appearsto be fine needles or fiber-like particles.

The monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis synthesized according to Example 1.

A particular embodiment of this invention is a crystalline form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,wherein the x-ray powder diffraction pattern (XRPD) contains thefollowing 2θ peaks measured using CuK_(α) radiation: 7.14, 8.89, 10.22,12.42, 12.73 and 14.31. Details on the method of obtaining the XRPDcalculations are provided in Example 1. Another embodiment of theinvention is a pharmaceutical formulation comprising the crystallineform of the Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand a pharmaceutically acceptable excipient.

Another embodiment of this invention relates to the other crystal formsof the Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein other solvents, including ethanol, acetone, acetonitrile, and mixedsolvents. Details on the other forms of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand the method of obtaining the XRPD calculations are provided inExample 4.

The invention also relates to a composition comprising the monohydrateform of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,represented by Formula (I), and pharmaceutical formulation comprisingthe monohydrate form of the Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand a pharmaceutically acceptable excipient.

The pharmaceutical formulations of the Trk inhibitor may comprise one ormore pharmaceutically acceptable excipients. Modes of administration ofthe pharmaceutical formulations of the Trk inhibitor include oral,sublingual, intravenous, subcutaneous, intramuscular, intra-articular,transdermal, rectal, inhalation, intrathecal/intraventricular, andtopical. Accordingly, the pharmaceutical formulations of the Trkinhibitor may be formulated, for example, as a capsule, tablet, powder,solution, suspension, emulsion, lyophilized powder, or an extendedrelease formulation comprising injectable microcapsules. The excipientsused in the pharmaceutical formulations of the Trk inhibitor will dependon the route of administration for which the pharmaceutical formulationof the Trk inhibitor is intended.

Suitable excipients include, but are not limited to, inorganic ororganic materials such as diluents, solvents, gelatin, albumin, lactose,starch, stabilizers, melting agents, emulsifying agents, suspendingagents, salts and buffers. Suitable pharmaceutically acceptableexcipients for intra-articular formulations such as solutions orsuspensions include, but are not limited to, commercially availableinert gels or liquids.

Given the low solubility of the Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,a suspending agent is needed to make a microcrystalline suspensionformulation for intra-articular injection comprising the Trk inhibitoras the active pharmaceutical ingredient Commonly used pharmaceuticallyacceptable suspending agents include: acacia, agar, alginic acid,bentonite, calcium stearate, carbomer, carboxymethylcellulose (calciumand sodium), carrageenan, cellulose (microcrystalline, microcyrstallineand carboxymethylcellulose sodium, powdered), colloidal siliconedioxide, destrin, gelatin, guar gum, hectorite, hydrophobic colloidalsilica, hydroxyethyl cellulose, hydroxymethyl celluslose, hydroxypropylcellulose, hypromellose, kaolin, magnesium aluminum silicate, maltitolsolutions, medium-chain triglycerides, methylcellulose, phenylmercuricborate, phospholipids, poycarbophil, polyethylene glycol,polyoxyethylene sorbitan fatty acid esters, povidone(polyvinylpyrrrolidone), propylene glycol alginate, saponite, sesameoil, sodium alginate, sorbitan esters, sucrose, tragacanth, vitamin Epolyethylene glycol succinate, and xanthan gum (Handbook ofPharmaceutical Excipients, 6^(th) Edition).

Buffering agents are also used in the formulation of a solution forintra-articular administration where the active ingredient is themonohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,the Trk inhibitor. Pharmaceutically acceptable buffering agents include:adipic acid, ammonia solution, boric acid, calcium carbonate, calciumhydroxide, calcium lactate, calcium phosphate, tribasic, citric acidmonohydrate, dibasic sodium phosphate, diethanolamine, glycine, maleicAcid, malic acid, methionine, monobasic sodium phosphate,monoethanolamine, monosodium glutamate, phosphoric acid, potassiumcitrate, sodium acetate, sodium bicarbonate, sodium borate, sodiumcarbonate, sodium citrate dihydrate, sodium hydroxide, sodium lactate,and triethanolamine (Handbook of Pharmaceutical Excipients, 6^(th)Edition).

As the pharmaceutical formulations of the Trk inhibitor of the instantinvention are formulations for intra-articular administration, they alsomay contain diluents. Suitable diluents for applications as in theinstant invention include: malitol, sunflower oil, ammonium alginate,calcium carbonate, calcium lactate, calcium phosphatedibasic anhydrous,dibasic dihydrate, tribasic, calcium silicate, calcium sulfate,cellulose (powdered, silicified microcrystalline), cellulose acetate,compressible sugar, confectioner's sugar, corn starch and pregelatinizedstarch, dextrates, dextrin, dextrose, erythritol, ethylcellulose,fructose, fumaric acid, glyceryl palmitostearate, inhalation lactose,isomalt, kaolin, lactitol, lactose (anhydrous, monohydrate and cornstarch, monohydrate and microcrystalline cellulose, spray dried),magnesium carbonate, magnesium oxide, maltodextrin, maltose, mannitol,medium-chain triglycerides, microcrystalline cellulose, polydextrose,polymethacrylates, simethicone, sodium alginate, sodium chloride,sorbitol, starch (pregelatinized, sterilizable maize), sucrose, sugarspheres, sulfobutylether b-cyclodextrin, talc, tragacanth, trehalose,xylitol (Handbook of Pharmaceutical Excipients, 6th Edition).

Microcrystalline suspension pharmaceutical formulations of the Trkinhibitor with the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineas the active ingredient are described in Example 2.

This invention also relates to methods of manufacturing a crystallineform of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand manufacturing the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminecomprising:

-   -   a. Mixing        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        with a solvent to form a suspension;    -   b. Stirring the suspension;    -   c. Collecting the solids in the suspension by filtration; and    -   d. Drying the solids.        In this method, the solvent used to form the suspension may be a        mixture of acetone and water. Further, the suspension may be        stirred overnight, and the solids that are collected may be air        dried.

Extended release pharmaceutical formulations of the Trk inhibitor maycomprise either3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineor the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineas, in the case of formulations produced from drug solutions, the Trkinhibitor is dissolved in solvent prior to microencapsulation andformulation, and in the case of formulations produced from drugsuspensions, the Trk inhibitor is suspended in solvent prior tomicroencapsulation and formulation. A solution or suspension containingthe active ingredient is then combined with various polymers, as setforth in the Example 3, in order to microencapsulate the3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.The microencapsulation of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminehelps to provide a formulation with an extended effect, up to and over 3months in duration, and to provide sustained therapeutic effect in thepatient.

In a particular embodiment of the invention, the pharmaceuticalformulation comprising the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminecomprises a pharmaceutically acceptable diluent, a pharmaceuticallyacceptable suspending agent and a pharmaceutically acceptable bufferingagent. In certain embodiments of the pharmaceutical formulations of theinstant invention, the diluent is sorbitol, the suspending agent ispovidone, and the buffering agent is phosphoric acid.

The instant invention also relates to extended release pharmaceuticalformulations comprising3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrospheres. In these extended release formulations, the Trk inhibitormay be either3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineor the monohydrate form of the compound. The drug-loaded microspheresmay further comprise a polymer.

In a specific embodiment of a Trk inhibitor-loaded microspherecomprising a polymer, the polymer is selected from poly(D,L-lactide),poly(D,L-lactide-co-glycolide) and a combination of bothpoly(D,L-lactide) and poly(D,L-lactide-co-glycolide). In certainembodiments of the invention, the poly(D,L-lactide) used in the extendedrelease formulation has an inherent viscosity of 0.16-0.35 dL/g. Inanother embodiment, poly(D,L-lactide) has an inherent viscosity of0.16-0.24 dL/g. In yet another embodiment, the poly(D,L-lactide) has aninherent viscosity of 0.25-0.35 dL/g. In certain embodiments of theinvention, the poly(D,L-lactide-co-glycolide) has an inherent viscosityof 0.14-0.24 dL/g. In one embodiment, poly(D,L-lactide-co-glycolide) hasan inherent viscosity of 0.14-0.22 dL/g. In another embodiment thepoly(D,L-lactide-co-glycolide) has an inherent viscosity of 0.16-0.24dL/g.

In an extended release formulation comprising both poly(D,L-lactide) andpoly(D,L-lactide-co-glycolide), the poly(D,L-lactide) has an inherentviscosity of 0.16-0.35 dL/g and the poly(D,L-lactide-co-glycolide) hasan inherent viscosity of 0.14-0.24 dL/g. In one embodiment, thepoly(D,L-lactide) has an inherent viscosity of 0.16-0.24 dL/g and thepoly(D,L-lactide-co-glycolide) has an inherent viscosity of 0.14-0.22dL/g. In another embodiment, the poly(D,L-lactide) has an inherentviscosity of 0.16-0.24 dL/g and the poly(D,L-lactide-co-glycolide) hasan inherent viscosity of 0.16-0.24 dL/g. In yet another embodiment, thepoly(D,L-lactide) has an inherent viscosity of 0.25-0.35 dL/g and thepoly(D,L-lactide-co-glycolide) has an inherent viscosity of 0.14-0.22dL/g. And in yet another embodiment, the poly(D,L-lactide) has aninherent viscosity of 0.25-0.35 dL/g and thepoly(D,L-lactide-co-glycolide) has an inherent viscosity of 0.16-0.24dL/g.

In another embodiment, the extended release pharmaceutical formulationscomprising Trk inhibitor-loaded microspheres comprise a 9:1 ratio ofpoly(D,L-lactide) and poly(D,L-lactide-co-glycolide). In these extendedrelease formulations, the poly(D,L-lactide) has an inherent viscosity of0.16-0.35 dL/g and the poly(D,L-lactide-co-glycolide) has an inherentviscosity of 0.14-0.24 dL/g.

In another embodiment, the extended release pharmaceutical formulationscomprising Trk inhibitor-loaded microspheres comprise a 9.5:0.5 ratio ofpoly(D,L-lactide) and poly(D,L-lactide-co-glycolide). In theseformulations, the poly(D,L-lactide) has an inherent viscosity of0.16-0.35 dL/g and the poly(D,L-lactide-co-glycolide) has an inherentviscosity of 0.14-0.24 dL/g.

In the extended release pharmaceutical formulation comprising Trkinhibitor-loaded microspheres, the microspheres are loaded with 1% w/wto 99% w/w3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.In a more specific embodiment, the microspheres are loaded with 12% w/wto 50% w/w3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.In yet another embodiment, the microspheres are loaded with 12% w/w to50% w/w3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.In more specific embodiments, the microspheres are loaded with 12% w/w,15% w/w, 16% w/w, 17% w/w, 18% w/w, 19% w/w, 20% w/w, 25% w/w, 30% w/w,40% w/w or even 50% w/w3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.

This invention also relates to methods of manufacturing3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrocapsules by solvent extraction and by spray drying.

One solvent extraction method of the instant invention relates toforming Trk inhibitor-loaded microspheres from a solution of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.This method comprises:

-   -   a. Dissolving the        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        in an organic solvent to form a drug solution;    -   b. Adding a polymer to the drug solution to form a        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        solution;    -   c. Mixing the        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        solution into an aqueous solution to form an emulsion;    -   d. Adding deionized water to the emulsion;    -   e. Forming microspheres from the emulsion by solvent extraction;        and f. Sieving the resulting microspheres using a surfactant        solution.

In this method, the organic solvent may comprise (i) dichloromethane andmethanol, (ii) dichloromethane, (iii) benzyl alcohol and methanol, (iv)dichloromethane and benzyl alcohol, (v) choloroform, (v) chloroform andmethanol, or (vii) chloroform and benzyl alcohol. The polymer in thismethod may be poly(D,L-lactide), poly(D,L-lactide-co-glycolide), or acombination of poly(D,L-lactide) and poly(D,L-lactide-co-glycolide). Theaqueous solution in this method may be polyvinyl alcohol in water. Thesurfactant solution in this method may be poloxamer 407 in water,polysorbate 80 in water, or polysorbate 20 in water.

Another solvent extraction method of the instant invention relates toforming Trk inhibitor-loaded microspheres from a suspension of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.This method comprises:

-   -   a. Dispersing the        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        in an organic solvent to form a drug suspension;    -   b. Adding a polymer to the drug suspension to form a        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        dispersion;    -   c. Mixing the        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        dispersion with an aqueous solution to form an emulsion;    -   d. Adding deionized water to the emulsion;    -   e. Forming microspheres from the emulsion by solvent extraction;        and    -   f. Sieving the resulting microspheres using a surfactant        solution.

In this method, the organic solvent may comprise (i) ethyl acetate, (ii)dichloromethane, (iii) chloroform, (iv) ethyl acetate anddichloromethane, (v) ethyl acetate and chloroform, (vi) dichloromethaneand chloroform or (vii) ethyl acetate, dichloromethane and chloroform.The polymer in this method may be poly(D,L-lactide),poly(D,L-lactide-co-glycolide), or a combination of poly(D,L-lactide)and poly(D,L-lactide-co-glycolide). The aqueous solution in this methodmay be polyvinyl alcohol in water. The surfactant solution in thismethod may be poloxamer 407 in water, polysorbate 80 in water, orpolysorbate 20 in water.

In another aspect, the invention relates to a method of forming Trkinhibitor-loaded microspheres from a solution of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrocapsules by spray drying. This method comprises:

-   -   a. Dissolving the        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        in an organic solvent to form a drug solution;    -   b. Adding a polymer to the drug solution to form a        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        solution; and    -   c. Pumping the        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        solution through a sprayer into a dryer to form a spherical        particle.

In this method, the organic solvent may comprise (i) dichloromethane andmethanol, (ii) dichloromethane, (iii) benzyl alcohol and methanol, (iv)dichloromethane and benzyl alcohol, (v) choloroform, (v) chloroform andmethanol, or (vii) chloroform and benzyl alcohol. The polymer in thismethod may be poly(D,L-lactide), poly(D,L-lactide-co-glycolide), or acombination of poly(D,L-lactide) and poly(D,L-lactide-co-glycolide).Additional parameters of this method relate to the spray rate andatomizing nitrogen flow of the sprayer. The spray rate may be 0.7mL/min; the atomizing nitrogen flow may be 4 L/min. The temperature atvarious points in the dryer are also elements that may be controlled toimpact the resulting microsphere size. In this method, the dryer mayhave an inlet temperature of 50° C., a chamber temperature of 40-45° C.,and an exhaust temperature of 20-30° C. In a more specific embodiment,the chamber temperature is 40-43° C.; more specifically the chambertemperature is 41-43° C. In another embodiment, the the exhausttemperature is 22-28° C.

In yet another aspect, the invention relates to a method of forming Trkinhibitor-loaded microspheres from a suspension of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrocapsules by spray drying. This method comprises:

-   -   a. Dispersing the        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        in an organic solvent to form a drug suspension;    -   b. Adding a polymer to the drug suspension to form a        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        dispersion; and    -   c. Pumping the        polymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        dispersion through a sprayer into a dryer to form a spherical        particle.

In this method, the organic solvent may comprise (i) ethyl acetate, (ii)dichloromethane, (iii) chloroform, (iv) ethyl acetate anddichloromethane, (v) ethyl acetate and chloroform, (vi) dichloromethaneand chloroform or (vii) ethyl acetate, dichloromethane and chloroform.The polymer in this method may be poly(D,L-lactide),poly(D,L-lactide-co-glycolide), or a combination of poly(D,L-lactide)and poly(D,L-lactide-co-glycolide). Additional parameters of this methodrelate to the spray rate and atomizing nitrogen flow of the sprayer. Thespray rate may be 0.7 mL/min; the atomizing nitrogen flow may be 4L/min. The temperature at various points in the dryer are also elementsthat may be controlled to impact the resulting microsphere size. In thismethod, the dryer may have an inlet temperature of 50° C., a chambertemperature of 40-45° C., and an exhaust temperature of 20-30° C. In amore specific embodiment, the chamber temperature is 40-43° C.; morespecifically the chamber temperature is 41-43° C. In another embodiment,the exhaust temperature is 22-28° C.

The following non-limiting Examples illustrate the various embodimentsof the invention, including methods for preparing the monohydrate formof3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,pharmaceutical formulations with the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,extended release pharmaceutical formulations of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand methods for producing these extended release formulations.

EXAMPLES Example 1: The Monohydrate Form of3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineExample 1-1: Synthesis of the Monohydrate Form of3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine

To a stirred solution of3-methoxy-4-((4-methoxybenzyloxy)phenyl)methanamine (2.00 g, 7.32 mmol)and 5-bromo-2-chloro-3-nitropyridine (1.66 g, 6.97 mmol) in acetonitrile(50 mL) was added N,N-diisopropylethylamine (1.13 g, 8.71 mmol). Theresulting mixture was heated to reflux and allowed to stir. After 64 h,the reaction mixture was allowed to cool to room temperature and wasdiluted with water. The mixture was extracted twice withdichloromethane. The combined organic phases were dried over magnesiumsulfate, filtered, and concentrated to provide 3.34 g (>100%) of5-bromo-N-(3-methoxy-4-((4-methoxybenzyloxy)benzyl)-3-nitropyridin-2-amineas a yellow-brown solid.

To a stirred solution of5-bromo-N-(3-methoxy-4-((4-methoxybenzyloxy)benzyl)-3-nitropyridin-2-aminein tetrahydrofuran (40 mL), ethanol (40 mL), and water (40 mL) was addedsodium hydrosulfite (6.09 g, 34.99 mmol). The resulting mixture washeated to reflux and allowed to stir. After 4 h, the reaction mixturewas allowed to cool to room temperature and was diluted with water. Theyellow mixture was extracted three times with dichloromethane. Thecombined organic phases were washed with brine, dried (magnesiumsulfate), filtered, and concentrated to provide 3.10 g of a yellow-brownsolid. Chromatographic purification (Combi-Flash 40 g SiO₂ gold column,1-2.5% methanol/dichloromethane) afforded 1.28 g (51%) of5-bromo-N²-(3-methoxy-4-((4-methoxybenzyloxy)benzyl)pyridine-2,3-diamineas a yellow solid.

To a stirred solution of5-bromo-N²-(3-methoxy-4-((4-methoxybenzyloxy)benzyl)pyridine-2,3-diamine(0.850 g, 1.91 mmol) in dichloromethane (30 mL) and methanol (30 mL) wasadded cyanogen bromide (5.0 M in acetontitrile, 573 μL, 2.87 mmol). Theresulting solution was allowed to stir at room temperature. After 24 h,a second aliquot of cyanogen bromide solution was added (600 μL) andstirring continued. After 48 h, a third aliquot of cyanogen bromidesolution (600 μL) was added and stirring continued. After a total of 72h, the reaction mixture was concentrated, and the residue was dissolvedin dichloromethane. The solution was washed with 1N sodium hydroxidesolution, dried over magnesium sulfate, filtered, and concentrated toprovide 1.17 g of a brown solid. Chromatographic purification(Combi-Flash, 40 g SiO₂ gold column, 1-10% 2M ammonia inmethanol/dichloromethane) afforded 0.28 g (32%) of6-bromo-3-(3-methoxy-4-((4-methoxybenzyloxy)benzyl)-3H-imidazo[4,5-b]pyridin-2-amineas a brown solid.

To a stirred solution of6-bromo-3-(3-methoxy-4-((4-methoxybenzyloxy)benzyl)-3H-imidazo[4,5-b]pyridin-2-amine(0.25 g, 0.53 mmol) in 1,4-dioxane (10 mL) and water (4 mL) was added1-methyl-4-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole(0.14 g, 0.66 mmol), potassium phosphate tribasic (0.39 g, 1.84 mmol),tricyclohexylphosphine (0.015 g, 0.052 mmol), palladium(II) acetate(0.005 g, 0.026 mmol). The reaction mixture heated to 125° C. in amicrowave reactor. After 15 min, the reaction mixture was allowed tocool to room temperature and was diluted with water. The mixture wasextracted twice with ethyl acetate. The combined organic phases werewashed with brine, dried over magnesium sulfate, filtered, andconcentrated to provide 0.36 g of a greenish brown solid.Chromatographic purification (Combi-Flash, 12 g SiO₂ gold column, 1-10%2M ammonia in methanol/dichloromethane) afforded 0.10 g (41%) of theproduct as a light green solid.

3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewas added to a mixture of acetone (2 mL) and water (0.1 mL). Thesuspension was stirred by a magnetic stirring bar at room temperatureovernight. The solids were collected by filtration and dried in the air.The structure was confirmed by proton NMR. The differential scanningcalorimetry (DSC) thermogram exhibits three thermal events at 76.72,160.13, and 195.78° C., the thermal gravimetric analysis (TGA)thermogram shows 3.7% weight loss from 25-100° C., and the X-ray powderdiffraction analysis (XRPD) shows unique peaks at 3.6, 7.1, 8.9, 10.4,10.7, 12.4, 12.7 and 14.3 2θ peaks measured using CuK_(α) (accuracy±0.2°).

Example 1-2: Identification of the Crystalline Structure of theMonohydrate Form of3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine

The crystalline structure of the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewas also solved. Crystals of the monohydrate form of of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewere recrystallized from acetone/water. A crystal 0.20*0.08*0.03 mm insize yielded to suitable diffraction frames when it was placed on thetop of a MiTeGen MicroMount. X-ray diffraction data were collected on aBruker/AXS three circle diffractometer, equipped with a SMART APEXarea-detector, a low temperature device (model LT 2) and a copper-Kmicrofocus generator, operated at 45 kV/650 μA and a focusing beamMontel multilayer optic with an image focus spot diameter of ˜250 μm(Wiesmann et al., 2007). Data were collected using the program packageSMART V 5.628 (Bruker AXS, 2001), and processed with the programSAINT+Release 6.45 (Bruker AXS, 2003). This analysis yielded 2459reflections (ϑ_(min)=1.78, ϑ_(max)=50.21; 0<h<24, −4<k<0, −19<1<19) ofwhich all 2459 reflections were unique (R_(int)=n.a., R_(σ)=0.1583).Refinement of the cell parameters was performed using 1405 reflections.An empirical absorption correction was applied and the phase problem wassolved with the “structure-solution” module of the APEX2 suite.

The structure was refined by least-squares methods (minimization of(F_(o) ²−F_(c) ²)²) using the XL module of the APEX2 suite (Bruker AXS,2011). The positions of all hydrogen atoms were calculated,S_(goodness of fit)=1.085, R_(all data)=0.1329 (R_(obs. data)=0.0920 for1614 reflections with |F_(obs)|>4σ, wR2_(all data)=0.2710,wR2_(obs. data)=0.2377). The largest unassigned peaks in the differencemap correspond to −0.348 versus +0.386 electrons per Å³. The averageestimated standard deviation (e.s.d.) of a C—C bond is 0.009 Å, that ofan O—C bond is 0.009 Å and that of a N—C bond is 0.009 Å. The averagee.s.d. of C—C—C bond angles is 0.7 and that of C—C—C—C torsion angles1.004°.

The crystalline structure of the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis presented in FIG 3. FIG. 4 shows an overlay of the experimentalpowder pattern with the one calculated from the single crystalstructure. The strong degree of matching suggests that the singlecrystal structure is indicative of the bulk material.

Example 2:3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineMonohydrate Microcrystalline Suspension Formulations

Microcrystalline suspension pharmaceutical formulations of the Trkinhibitor, where the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis the active ingredient, were developed. These formulations weredeveloped as a suspension of the insoluble3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineparticles to sustain the delivery of the Trk inhibitor to the body overtime. This approach relies on the poor solubility of the activeingredient and dose to control the duration of release; it also showssustained delivery of approximately 1 month of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein vivo.

Example 2-1: 20 mg/mL3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineMonohydrate Microcrystalline Suspension for Injection

A pharmaceutical formulation comprising 20 mg of the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineper mL of solution was prepared. The composition of the 20 mg/mLmicrocrystalline suspension is provided in Table 1 below.

TABLE 1 20 mg/mL Microcrystalline Suspension Formulation CompositionComponent per Unit Function Monohydrate form of 3-(3-methoxy- 72 mgActive 4-((4-methoxybenzyl)oxy)benzyl)-6- ingredient(1-methyl-1H-pyrazol-4-yl)-3H- imidazo[4,5-b]pyridin-2-amine Sorbitol162 mg Diluent Povidone K17 pyrogen free 72 mg Suspending agentPhosphate buffer (10 mM, pH 7.4) 3.6 mL^(†) Buffering agent ^(†)Anoverfilling of 10% is applied

Example 2-2: Method of Manufacturing the 20 mg/mL3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineMonohydrate Microcrystalline Suspension Formulation

The 20 mg/mL solution of the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewas prepared according to the following steps:

-   -   1. A phosphate buffer 10 mM pH 7.4 is prepared by combining        appropriate amounts of Water for Injection (WFI) and sodium        dihydrogenophosphate, stirring to complete dissolution, and then        adjusting the pH to 7.4 (±0.2) with sodium hydroxide 1N        solution.    -   2. A vehicle is prepared by combining the appropriate amounts of        sorbitol and Povidone K17 pyrogen, adding the phosphate buffer        10 mM pH 7.4 and stirring until complete dissolution.    -   3. The resulting vehicle is then filtered through a 0.22 μm PVDF        [polyvinylidene fluoride] hydrophilic filter.    -   4. A concentrated suspension is obtained by mixing the        monohydrate form of        3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine        with approximately the vehicle to obtain a 30 mg/mL concentrated        suspension.    -   5. The concentrated suspension, kept under agitation, is        filtered on a 75 μm stainless steel sieve.    -   6. The pre-filtered suspension remains under agitation and is        filtered on a 40 μm stainless steel sieve to obtain the        concentrate suspension.    -   7. The concentrate suspension is adjusted with the filtered        vehicle to obtain a 20 mg/mL suspension.    -   8. The final suspension is filled, into sterile and        depyrogenated colorless type I glass vials to a 3.6 mL filling        volume. Vials are closed with sterile and depyrogenated ETFE        coated bromo-butyl stoppers. Stoppers are crimped with sterile        aluminum caps and sterile white plastic lids onto the vials.    -   9. The filled vials are sterilized in autoclaving equipment.

Example 2-3: Clinical Doses of the 20 mg/mL3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineMonohydrate Microcrystalline Suspension Formulation

The 20 mg/mL microcrystalline suspension of the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineis further modified to achieve the dose and volume to be administered tothe patient in the finished drug product configurations. The targetdoses are reconstituted as necessary from the 20 mg/mL microcrystallinesuspension. A summary of the various dosage and volume formats areprovided in Table 2.

TABLE 2 Clinical Presentations of the 20 mg/mL 3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine Monohydrate Microcrystalline SuspensionTargeted Total Volume of Final Concentration Volume Dose ReconstitutedVial in Vial Administered to (mg) (mL) (mg/mL) Patient (mL) 3 3.47 0.983.2 10 3.95 3.29 3.2 30 6.60 10 3.2 60 n/a^(†) 20 3.2 100 n/a^(†) 205.4^(‡) ^(†)Reconstitution not required to achieve desired finalconcentration. ^(‡)2 × 2.7 mL vials

Example 3:3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineExtended Release Formulations

In some cases, the duration of drug release is desired to be extended,for example to greater than 3 month exposure time. Accordingly, extendedrelease formulations of the Trk inhibitor3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineas the active ingredient are produced and formulated using either theanhydrate or monohydrate form of the compound. The extended releaseformulation approach uses poly(D,L-lactide) (PLA),poly(D,L-lactide-co-glycolide) (PLGA) polymers or a combination ofPLA-PLGA polymers to encapsulate3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine(the active pharmaceutical ingredient [API] or drug substance [DS]) toproduce a drug product [DP] solution of injectable microcapsules. Theseformulations can provide sustained, or greater than 3 months, exposureof the drug to the body. A summary of the different polymers used in thepreparation of the extended release formulations of this example isprovided in Table 3.

TABLE 3 Polymers Used in3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine Monohydrate Extended ReleaseFormulations Evonik Polymer Inherent Reference Viscosity* Code PolymerName General Structure (g/mol) End Group R202H Poly(D,L-lactide) [PLA]

0.16-0.24 Acid R202S Poly(D,L-lactide) [PLA]

0.16-0.24 Ester R203H Poly(D,L-lactide) [PLA]

0.25-0.35 Acid R203S Poly(D,L-lactide) [PLA]

0.25-0.35 Ester RG502H Poly(D,L-lactide- co-glycolide) 50:50 [PLGA]

0.16-0.24 Acid RG752H Poly(D,L-lactide- co-glycolide) 75:25 [PLGA]

0.14-0.22 Acid *Inherent Viscosity is measured at 0.1% w/v in CHCl₃ at25° C., with a Ubbelhode size 0c glass capillary viscometer. Source:http://www.resomer.com/product/biodegradable-polymers/en/pharma-polymers/products/pages/bioresorbable-polymer.aspx

The extended release pharmaceutical formulations of Trk inhibitors with3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineas the active ingredient use bioerodible polymers, poly(D,L-lactide)and/or poly(D,L-lactide-co-glycolide), to alter the control of releaseof the active ingredient from drug particle dissolution to polymerhydrolysis. Using the appropriate combination of polymers and activeingredient loading, the drug release rate can be controlled to result in3 or more months of exposure. Particular combinations of PLGA/PLApolymers and3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineare combined in a manner that extends the duration of drug release togreater than 3 months; these formulations are discussed further in thisexample.

Example 3-1: Effect of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineLoading on In Vitro Release Profile

Pharmaceutical compositions comprising microspheres of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewere prepared by solvent extraction. The amounts of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand PLA/PGLA polymer masses used to prepare each batch are presented inTable 4. The in vitro release (IVR) kinetics of these variousformulations were examined.

TABLE 4 Compositions of Microspheres^(‡)-12% API/9:1 R202H:752H, 16%API/9:1 R202H:752H and 20% API/9:1 R202H:752H GZ389988^(†) GZ389988Polymer Polymer Batch# Loading weight (mg) Ratio Amount (mg)* 1 12% 369:1 234:26 R202H:752H 2 16% 50 9:1 234:26 R202H:752H 3 20% 65 9:1 234:26R202H:752H ^(‡)Prepared by solvent extraction ^(†)GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine*Total Polymer Amount = 260 mg

3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewas dissolved in 2 mL of 9:1 dichloromethane (DCM):methanol (MeOH) withgentle heating (˜30 minutes). Polymers were added and dissolved over 30minutes. Separately, 39 mL of cold, sterile-filtered polyvinylamine(PVA) solution (5% w/v in water) was placed in a 250 mL beaker equippedwith a 7.9×38.1 mm Teflon magnetic stir bar. The beaker was placed in anice bath on an IKA RCT basic stir plate set at 500 rpm. The polymer/drugsolution was filtered into the PVA solution using a Pall Acrodisc 0.2 μmPTFE syringe filter attached to a 5 cc glass Hamilton syringe. Uponaddition, the polymer/drug solution formed an emulsion. After 1 minute,160 mL of cold deionized water was added. After 5 minutes, the stirspeed was decreased to 300 rpm. The microspheres were formed by solventextraction over 3 hours. Microspheres were sieved through 75 μm and 38μm stacked sieves using cold 0.1% Kolliphor P 407 in deionized water;the 38-75 μm fraction was collected and the excess rinse solution wasremoved. The microspheres were frozen at −80° C. and lyophilized.

The IVR kinetics of the release of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminefrom the microsphere formulations was determined by preparing a 2% (w/v)aqueous suspension of microspheres in 0.2% hyaluronic acid+0.2%Kolliphor P 407, intended to mimic the intra-articular environment ofthe knee. In triplicate, volumes of the suspensions containing atheoretical loading of 500 μg3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewere pipeted into separate 50 mL glass centrifuge tubes containing 0.5%sodium dodecyl sulfate in PBS, pH 7.4 release media. The tubes wereplaced on their side in a reciprocal shaker incubator at 37° C. At eachtimepoint, the microspheres were allowed to settle and 1 mL of releasemedia was sampled and replaced. To determine the actual total mass of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein the IVR samples, the same volumes of suspensions used for the releasestudy were sampled (in triplicate) and 10 mL dimethyl sulfoxide wasadded. The samples were sonicated, gently heated and placed on a rockerat room temperature to dissolve. These samples were analyzed for total3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminecontent using HPLC-UV. A cumulative IVR profile was plotted aspercentage of actual3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineloading versus time.

3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineencapsulation efficiency was determined by dividing the actual drugloading by the theoretical loading. To determine the actual loading,accurately weighed masses of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineloaded microspheres were dissolved in dimethyl sulfoxide and the3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineconcentration was measured by HPLC-UV.

IVR profiles showed that drug loading levels above 16% (w/w) showed aburst release (FIG. 5). The cause for this burst was identified bydifferential scanning calorimetry (DSC) and scanning electron microscopy(SEM). DSC analysis indicated that drug loading levels above 16% (w/w)showed drug crystallization in the microspheres as evidenced by a meltendotherm at 130-150° C. (FIG 6.). Drug crystallization was alsorevealed by SEM, where drug loading levels above 16% showed drugcrystals on microsphere surfaces (FIG 7.)

FIG. 6 demonstrates that 20% microspheres show a melting endothermbetween 130-150° C. confirming the presence of surface drug crystals.FIG. 7 and FIG. 8 show SEMs of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrospheres at 1500×. FIG. 7, 16% drug-loaded microspheres, shows nodrug crystals (drug is amorphous); FIG. 8, 20% drug-loaded microspheres,shows surface drug crystals.

The effect of drug crystallization on IVR was also demonstrated usingrelated microsphere compositions. FIG. 9 shows the IVR of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminefrom microspheres prepared with R202H polymer. FIG. 10 shows the IVR of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminefrom microspheres prepared with a 9.5:0.5 ratio of R202H and RG752Hpolymers. In these examples, drug loading levels above 16% (w/w) showedburst release; the extent of drug burst was directly related to the drugloading level.

Example 3-2: Effect Poly-Lactide and Poly-Lactide-Co-Glycolide PolymerBlends on In Vitro Release Profile of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineExample 3-2-1: 16% API/R202H, 16% API/9.0:0.5 R202H:RG752H and 16%API/9:1 R202H:752H Microspheres

Pharmaceutical compositions comprising 16%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202H, 9.0:0.5 R202H:RG752H and 9:1 R202H:752H and 16%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202H, 9.0:0.5 R202H:RG502H and 9:1 R202H:RG502H were prepared toassess the effect of differing polymer blends on the in vitro releasekinetics of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.Table 5 presents the drug and polymer masses used to prepare each batch.The method used to prepare these formulations is described in Example3-1.

TABLE 5 3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2- amine MicrosphereFormulations with Poly-lactide and Poly-lactide-co-glycolide PolymerBlends (16% API/R202H, 16% API/9.0:0.5 R202H:RG752H and 16% API/9:1R202H:RG752H) GZ389988† GZ389988 weight Polymer Polymer Batch# Loading(mg) Ratio Amount (mg)* 4 16% 50 mg R202H 260 5 16% 50 mg 9.5:0.5 247:13R202H: RG752H 6 16% 50 mg 9:1 234:26 R202H: RG752H ‡Prepared by solventextraction †GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine*Total Polymer Amount = 260 mg

Encapsulation efficiency and IVR analyses were performed as described inExample 3-1. The encapsulation efficiency for formulations 4, 5, and 6were 96.1±2.6%, 87.8±6.3%, and 77.7±8.6% respectively.

Microspheres prepared using 16%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202H polymer exhibited a biphasic release rate, with a slow initialrelease rate from 0-70 days followed by a faster secondary release ratefrom 70-110 days (FIG. 11). The blending of a more hydrophilic polymer,RG752H (75:25 PLGA), into the PLA microsphere formulations increased theinitial rate of release. This PLGA polymer allowed faster water uptakeinto the microspheres leading to the rate increase. At the ratio of 9:1R202H:RG752H, the initial release matched the secondary release rate,producing a pseudo zero-order release profile over 3 months (FIG. 11).

Example 3-2-2: 16% API/R202H, 16% API/9.5:0.5 R202H:RG502H and 16%API/9:1 R202H:RG502H Microspheres

Pharmaceutical compositions comprising 16%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202H, 9.0:0.5 R202H:RG502H and 9:1 R202H:RG502H were prepared toassess the effect of differing polymer blends on the in vitro releasekinetics of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.Table 6 presents the drug and polymer masses used to prepare each batch.The method used to prepare these formulations is described in

Example 3-1.

TABLE 6 Compositions of Microspheres^(‡)-16% API/R202H, 16% API/ 9.5:0.5R202H:RG502H and 16% API/9:1 R202H:RG502H GZ389988 Polymer GZ389988†weight Amount Batch# Loading (mg) Polymer Ratio (mg)* 7 16% 50 mg R202H260 8 16% 50 mg 9.5:0.5 247:13 R202H:RG502H 9 16% 50 mg 9:1 234:26R202H:RG502H ^(‡)Prepared by solvent extraction †GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine*Total Polymer Amount = 260 mg

Encapsulation efficiency and IVR analyses were performed as described in

Example 3-1. The encapsulation efficiency for formulations 7, 8, and 9were 96.1±2.6%, 91.7±3.3%, and 94.2±2.5% respectively.

Microspheres prepared using 16%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202H polymer exhibited a biphasic release rate, with a slow initialrelease rate from 0-70 days followed by a faster secondary release ratefrom 70-110 days (FIG. 12). The blending of a more hydrophilic 50:50PLGA polymer (RG752H) into the PLA microsphere formulations increasedthe initial rate of release. RG752H allowed faster water uptake into themicrospheres, leading to this rate increase. At the ratio of 9.5:0.5R202H:RG502H, the initial release matched the secondary release rate,producing a pseudo zero-order release profile over 6 months (FIG. 12).

Example 3-3: Preparation and Characterization of MicrosphereFormulations Showing Zero-Order In Vitro Release Kinetics Over 180 Days

Pharmaceutical compositions comprising 15%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein 9:1 R203H:RG752H and 16%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein 9.0:0.5 R202H:RG502H were prepared and analyzed for in vitro releasekinetics. Table 7 presents the drug and polymer masses used to prepareeach batch.

TABLE 7 Compositions of Microspheres^(‡)-15% API/9:1 R203H: RG502H and16% API/9.5.0.5 R202H:RG502H) GZ389988 Polymer GZ389988† weight AmountBatch# Loading (mg) Polymer Ratio (mg)* 10 15% 46 mg 9:1 234:26R203H:RG752H 11 16% 50 mg 9.5:0.5 247:13 R202H:RG502H ^(‡)Prepared bysolvent extraction †GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine*Total Polymer Amount = 260 mgEncapsulation Efficiency and IVR Analyses were Performed as Described in

Example 3-1. The encapsulation efficiency for formulations 10 and 11were 89.7±2.1% and 91.7±3.3% respectively.

Microspheres prepared using 15%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R203H:752H polymers at a ratio of 9:1 and 16%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202H:RG502H polymers at a ratio of 9.5:0.5 exhibited a pseudozero-order release profile over 6 months (FIG. 13).

Example 3-4: Effect of Co-Solvent Systems in Preparation on In VitroRelease Profile of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineExample 3-4-1: 16% API/9:1 R202H:RG752H as Prepared in 9:1 DCM:MeOH,9:0.5:0.5 DCM:MeOH:BA, 9:5 DCM:BA and 9:1 DCM:BA

Pharmaceutical compositions comprising 16%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineloaded microspheres in 9:1 R202H:RG752H were prepared using variousco-solvent solutions with differing ratios of the solventsdichloromethane (DCM), methanol (MeOH) and benzyl alcohol (BA). Theratios used were 9:1 DCM:MeOH, 9:0.5:0.5 DCM:MeOH:BA, 9.5:0.5 DCM:BA,and 9:1 DCM:BA. Refer to Table 8 for drug, polymers and co-solvents usedto prepare each batch. The method used to prepare these formulations isdescribed in

Example 3-1.

TABLE 8 Compositions of Microspheres^(‡) Prepared with VariousCo-Solvent Systems-16% API/9:1 R203H:RG752H GZ389988† GZ389988 weightPolymer Amount. Co-Solvent Batch# Loading (mg) Polymer Ratio (mg)*System 12 16% 50 mg 9:1 234:26 9:1 R202H:RG752H DCM:MeOH 13 16% 50 mg9:1 234:26 9:0.5:0.5 R202H:RG752H DCM:MeOH:BA 14 16% 50 mg 9:1 234:269.5:0.5 R202H:RG752H DCM:BA 15 16% 50 mg 9:1 234:26 9:1 R202H:RG752HDCM:BA ^(‡)Prepared by solvent extraction (various co-solvent systems)†GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine*Total Polymer Amount = 260 mg

Encapsulation efficiency and IVR analyses were performed as described inExample 3-1. The encapsulation efficiency for bathes 12, 13, 14, and 15were 77.7±8.6%, 94.9±2.0%, 90.5±2.6%, and 94.4±1.5% respectively. Benzylalcohol was chosen to incorporate into the co-solvent systems due to itsenhanced ability to solubilize3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.FIG. 14 and FIG. 15 show that benzyl alcohol can be used as part of aco-solvent system with dichloromethane or dichloromethane and methanolto produce microspheres without affecting the rates of release. This maybe useful in reducing potential recrystallization of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineduring microsphere production.

Example 3-4-2: Use of DCM:BA to Increase3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineLoading

Pharmaceutical compositions comprising 16% and 25%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein 9:1 R202H:RG752H, 30% and 40%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202H, and 25%, 30%, 40% and 50%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R203H were prepared using varying volumes of dichloromethane (DCM)and benzyl alcohol (BA). Refer to Table 9 for drug and polymer massesand solvent volumes used to prepare each batch. The method used toprepare these formulations is described in

Example 3-1.

TABLE 9 Compositions of Microspheres^(‡) Prepared with DCM:BA Co-SolventSystem-16% API/9:1 R202H:RG752H, 25% API/9:1 R202H:RG752H, 30%API/R202H, 40% API/R202H, 25% API/R203H, 30% API/R203H, 40% API/R203H,50% API/R203H Co-Solvent System [volume of solvent + GZ389988† GZ389988weight Polymer Amount volume of solvent Batch# Loading (mg) PolymerRatio (mg)* (mL)] 16 16%  50 mg 9:1 234:26 0.2 mL BA + R202H:RG752H 1.8mL DCM 17 25%  84 mg 9:1 234:26 0.2 mL BA + R202H:RG752H 1.8 mL DCM 1830% 112 mg R202H 260 0.26 mL BA + 1.2 mL DCM 19 40% 174 mg R202H 260 0.4mL BA + 1.2 mL DCM 20 40% 174 mg R202H 260 0.5 mL BA + 1.2 mL DCM 21 25% 84 mg R203H 260 0.2 mL BA + 1.8 mL DCM 22 30% 112 mg R203H 260 0.2 mLBA + 1.8 mL DCM 23 40% 174 mg R203H 260 0.4 mL BA + 1.2 mL DCM 24 40%174 mg R203H 260 0.5 mL BA + 1.2 mL DCM 25 50% 260 mg R203H 260 0.5 mLBA + 1.2 mL DCM 26 50%  26 mg R203H 260 0.61 mL BA +  1.2 mL DCM^(‡)Prepared by solvent extraction †GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine*Total Polymer Amount = 260 mg

Encapsulation efficiency and IVR analyses were performed as described inExample 3-1. The encapsulation efficiency for the formulations producedwith varying volumes of dichloromethane (DCM) and benzyl alcohol (BA)were:

Batch Encapsulation Efficiency 16 94.4 ± 1.5% 17 96.0 ± 1.8% 18 99.2 ±3.6% 19 94.0 ± 1.0% 20 100.5 ± 1.0%  21 91.2 ± 0.5% 22 90.2 ± 2.8% 2395.5 ± 6.8% 24 95.4 ± 3.1% 25 95.0 ± 0.8% 26 94.7 ± 1.7%

Benzyl alcohol was chosen to incorporate into the co-solvent systems dueto its enhanced ability to solubilize3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.FIG. 10 shows that benzyl alcohol can be used as part of a co-solventsystem with dichloromethane to produce microspheres with loadings ashigh as 50% (w/w). Some of the release profiles shown have minimal burstrelease and kinetics that should achieve 3-6 month duration. The volumeof benzyl alcohol used in the process effects the burst release of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.Reducing the amount of benzyl alcohol in the solvent system, while stillmaintaining the solubility of the API,3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine,in the polymer solution, reduces the burst effect. Increasing APIloading provides benefit by decreasing the amount of polymeradministered to the patient. This is expected to improve thebiocompatibility of the implant and reduce the potential for polymeraccumulation after repeated administrations. In addition, increased APIloadings should translate into lower per-unit manufacturing costs.

Example 3-5: Effect of Micronized Suspension Microencapsulation Processon in Vitro Release Profile of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine

Pharmaceutical compositions comprising 25%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine(monohydrate form) in 9:1 R202H:RG752H, 30% and 40%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein 9:1 R203H:RG752H, 25%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202H, 30%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R202S, and 30%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R203 S were prepared with the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine.Refer to

Volume and Ethyl GZ389988 Polymer Composition of Acetate GZ389988^(†)weight Amount Dispersion Volume Batch# Loading (mg) Polymer Ratio (mg)*Solution (mL) (mL) 27 25%  84 mg 9:1 234:26 39 mL of 5% PVA 2.6R202H:RG752H 28 25%  84 mg 9:1 234:26 39 mL of 5% PVA 3.25 R202H:RG752H29 25%  84 mg R203H 260 39 mL of 5% PVA 2.0 30 30% 112 mg 9:1 234:26 39mL of 5% PVA + 2.0 R203H:RG752H 2.5% Ethyl Acetate 31 40% 174 mg 9:1234:26 39 mL of 5% PVA + 2.0 R203H:RG752H 2.5% Ethyl Acetate 32 30% 112mg 9:1 234:26 39 mL of 5% PVA + 2.0 R203H:RG752H 2.5% Ethyl Acetate 3330% 112 mg 9:1 234:26 39 mL of 5% PVA + 2.3 R203H:RG752H 2.5% EthylAcetate 34 30% 112 mg R202S 260 39 mL of 5% PVA + 2.0 2.5% Ethyl Acetate35 30% 112 mg R203S 260 39 mL of 5% PVA + 2.0 2.5% Ethyl Acetate

for drug and polymer masses used to prepare each batch. Batches 27, 28and 29 were prepared using3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminefrom manufacturing bathes, while Batches 30, 31, 32, 33, 34 and 35 wereprepared using3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminethat was micronized using a microfluidization process to ensure theparticle size was less than 10 μm diameter. This example compares thedifference in microspheres obtained when the polymer is dispersed in apolyvinyl alcohol (PVA) solution with or without ethyl acetate (EA), aswell as API loading, polymer composition and varying polymer solutionconcentrations (by varying EA volume).

TABLE 10 Compositions of Microspheres^(‡) Prepared with MicronizedSuspension Microencapsulation Process - 16% API/9:1 R202H:RG752H, 25%API/9:1 R202H:RG752H, 30% API/R202H, 40% AP1/R202H, 25% API/R203H, 30%API/R203H, 40% API/R203H, 50% API/R203H Volume and Ethyl GZ389988Polymer Composition of Acetate GZ389988^(†) weight Amount DispersionVolume Batch# Loading (mg) Polymer Ratio (mg)* Solution (mL) (mL) 27 25% 84 mg 9:1 234:26 39 mL of 5% PVA 2.6 R202H:RG752H 28 25%  84 mg 9:1234:26 39 mL of 5% PVA 3.25 R202H:RG752H 29 25%  84 mg R203H 260 39 mLof 5% PVA 2.0 30 30% 112 mg 9:1 234:26 39 mL of 5% PVA + 2.0R202H:RG752H 2.5% Ethyl Acetate 31 40% 174 mg 9:1 234:26 39 mL of 5%PVA + 2.0 R202H:RG752H 2.5% Ethyl Acetate 32 30% 112 mg 9:1 234:26 39 mLof 5% PVA + 2.0 R202H:RG752H 2.5% Ethyl Acetate 33 30% 112 mg 9:1 234:2639 mL of 5% PVA + 2.3 R202H:RG752H 2.5% Ethyl Acetate 34 30% 112 mgR202S 260 39 mL of 5% PVA + 2.0 2.5% Ethyl Acetate 35 30% 112 mg R203S260 39 mL of 5% PVA + 2.0 2.5% Ethyl Acetate ^(‡)Prepared by solventextraction ^(†)GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine*Total Polymer Amount = 260 mg

SEM analyses showed that the microspheres produced by encapsulating asuspension of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewere spherical with a rough surface texture due to the presence of drugcrystals embedded in the surface (FIG. 16). Encapsulation efficiency andIVR analyses were performed as described in Example 3-1. Theencapsulation efficiency for batch numbers 30, 31, 32, 33, 34, and 35were 103.3±3.8%, 101.6±5.3%, 94.7±4.0%, 97.1±0.2%, 35.6±0.8% and62.5±3.0%, respectively. Encapsulation efficiency was not performed onbatches 27, 28 or 29.

The IVR profiles showed burst release ranging from 25% to 47%, followedby a lack of release after 4 days (FIG. 17). Batches were evaluated forup to 21 days.

Example 3-6: Effect of Microencapsulation Process on In Vitro ReleaseProfile of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine

Pharmaceutical compositions comprising 16% and 25%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein 9:1 R202H:RG752H and 25% and 30%3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein R203H were prepared using a 9:1 ratio of dichloromethane (DCM):benzylalcohol (BA). Refer to Table 11 for drug and polymer masses used toprepare each batch. The method used to prepare Batch 23 is described in

Example 3-1, Batch 2. Batch 24 was prepared by dissolving3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein 5.016 g of 9:1 DCM/MeOH with gentle sonication. The two polymers werethen added and dissolved.

TABLE 11 Compositions of Microspheres Prepared by Solvent Extraction andSpray Drying-16% API/9:1 R202H:RG752H and No API/9:1 R202H:RG752HGZ389988† GZ389988 weight Polymer Amount Microencapsulation Batch#Loading (mg) Polymer Ratio (mg)* Process 36 16%   50 mg 9:1 234:26Solvent extraction R202H:RG752H 37 16% 238.8 mg 9:1 1140:114 Spraydrying R202H:RG752H 38 16% 238.8 mg 9:1 1140:114 Spray dryingR202H:RG752H 39 16% 238.8 mg 9:1 1140:114 Spray drying R203H:RG752H†GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine*Total Polymer Amount = 260 mg for batch 23 and 1254 mg for batch 24

Spray drying was performed using a ProCepT, 4M8 unit equipped with a twofluid nozzle with a 0.4 mm opening. The spray drying conditions andparameters are listed below:

Batch# 37 38 39 GZ389988 Load (%, w/w) 16 16 16 Solvent (MethyleneChloride:Methanol (v/v) 90:10 90:10 90:10 PLGA Solution Concentration(%, w/v): 20 22.5 22.5 Spray Rate (mL/min): 0.7 0.7 0.7 AtomizingNitrogen Flow (L/min): 4 4 4 Spray Amount (g): 6.4 5.8 5.8 InletTemperature (° C.): 50 50 50 Chamber Temperature (° C.): 41-43 40.7 40.0Exhaust Temperature (° C.): 27.4 22.0 22.1 Nitrogen Flow (m³/min): 0.350.35 0.35 Transfer Tube Pressure (mBar): 31-32 31-32 32 Yield (%, w/w)72.8 76.7 51.6

SEM analyses showed that solvent extraction microspheres were sphericalwith a smooth surface texture and spray dried microspheres werespherical with some surface texture (FIG. 18 and FIG. 19). Encapsulationefficiency and IVR analyses were performed as described in Example 3-1.The encapsulation efficiencies for solvent extraction batch 36 and spraydrying batches 37 and 38 were 107.4±11.6%, 91.4±4.3%, and 92.6±3.6%,respectively.

The two microencapsulation processes produced microspheres withnear-zero-order release profiles. FIG. 18 and FIG. 19 show SEMs of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrospheres at 1000×. The IVR profiles show different release rates(FIG. 20); the spray dried formulation achieves the desired duration ofdelivery of 3-6 months.

Example 3-7: Effect 1% 10 kDa PEG or 1% Poloxamer 407 on in vitroRelease Profile of Spray Dried Microspheres of 16% (w/w)3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine

This experiment was performed to determine if the addition of ahydrophilic additive can be used to accelerate the in vitro release rateof GZ389988 from PLA/PGA microspheres. In this experiment, microspheres(no additives) were compared with microspheres prepared with either 1%10 kDa PEG or 1% Poloxamer 407. Table 12 details the drug and polymermasses used to prepare each batch.

TABLE 12 Compositions of Microspheres^(‡) Prepared with 1% 10 kDa PEG or1% Poloxamer 407-16% API/1:1 R202H:R203H/No Additive, 16% API/1:1R202H:R203H/31.25 mg PEG, 16% API/1:1 R202H:R203H/31.25 mg Poloxamer 407GZ389988† GZ389988 weight Polymer Amount Additive Amount Batch# Loading(mg) Polymer Ratio (mg:mg) (mg) 40 16% 500 mg 1:1 1310:1310 N/AR202H:R203H 41 16% 500 mg 1:1 1297:1297 31.25 R202H:R203H 10 kDa PEG 4216% 500 mg 1:1 1297:1297 31.25 R202H:R203H Poloxamer 407 ^(‡)Prepared byspray drying †GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine

Spray drying was performed using a ProCepT, 4M8 unit equipped with abi-fluid nozzle with a 0.4 mm opening. The spray drying conditions andparameters are listed below:

Batch# 40 41 42 GZ389988 Load (%, w/w) 16 16 16 PLGA SolutionConcentration (%, w/v): 22.5 22.5 22.5 Spray Rate (mL/min): 0.7 0.7 0.7Air Flow (L/min): 0.35 0.35 0.35 Atomizing Nitrogen Flow (L/min): 4 4 4Chiller Temperature (° C.): −4 −4 −4 Inlet Temperature (° C.): 50 50 50Chamber Temperature (° C.): 40 40 40 Exhaust Temperature (° C.): 29 2929 Pre-Cyclone Pressure (mBar): 27-30 27-30 27-30

SEM analyses showed that spray dried microspheres prepared with orwithout hydrophilic additives showed a similar size distribution andsurface texture; all spray dried microspheres were spherical with somesurface texture (FIG. 21, FIG. 22, and FIG. 23). Encapsulationefficiency and IVR analyses were performed as described in Example 3-1.The encapsulation efficiencies for spray dried batch numbers 40, 41 and42 were 102.0±16.3%, 101.7±16.3% and 100.5±16.1%, respectively.

Microspheres prepared without additives showed slow, near zero-orderrelease of GZ389988 with approximately 20% of the active compoundreleased over 35 days (FIG. 24). The addition of either 1% 10 kDa PEG or1% Poloxamer 407 increased the release rate; approximately 42% of theactive compound was released over 35 days. This example demonstratesthat incorporation of a hydrophilic excipient accelerated the in vitrorelease rate of GZ389988 from PLA/PGA microspheres.

Example 4: In Vivo Studies Example 4-1: In vivo Performance of[¹⁴C]-3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine—LoadedMicrospheres Following Intra-Articular Injection into a Rat Knee Joint

Two formulations were selected to compare the in vitro release kineticswith the in vivo drug persistence in the rat knee joint. Theformulations are presented in Table 13.

In vitro release testing was performed using the method described in

Example 3-1. In vivo drug persistence testing was performed using[¹⁴C]-3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amineand unlabeled3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine;the two drug forms were co-solubilized during the microencapsulationprocess to ensure uniform drug distribution in the microspheres. Thetotal amount of radioactivity administered to each rat joint was ˜1.2MBq.

Evaluation of drug remaining in the rat knee joints was performed bysacrificing 2-3 rats at each time point, cryomilling the knee joint andextracting the3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminefrom the milled tissue. Quantitation was performed by liquidscintillation counting. The amount of radioactivity recovered in thejoint was calculated by dividing the radioactive counts at each timepoint by the radioactive counts in rats sacrificed at 0.1 hour followingtreatment. The concentration of radioactive drug in rat blood (expressedas nEq/g) was plotted over time and compared to the known IC50 value forthe compound.

TABLE 13 Compositions of Microspheres Tested In vivo-16% API/9:1202:H:RG502H and 15% API/9:1 R203H:RG752H GZ389988† + [14C] GZ389988GZ389988 weight Polymer Batch# Loading (mg) Polymer Ratio Amount (mg)*43 16% 50 mg 9:1 234:26 R202H:RG502H 44 15% 47 mg 9:1 234:26R203H:RG752H †GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine*Total Polymer Amount = 260 mg

The IVR profiles for batches 40 and 41 show near-zero-order release overapproximately 3-4 months and 5-6 months, respectively (FIG. 25).Following intra-articular administration into rat knee joints, Batches43 and 44 showed drug release over 5 to 6 months; Batch 43 showed 12%remaining in the joint after 5 months and Batch 44 showed 30% of thedrug remaining after 6 months (FIG. 26). The in vivo drug release ratewas slightly lower compared with the IVR rate likely due to localizationof the microspheres in the synovium.

FIG. 27 shows the drug concentration-time profile in blood. Followingintra-articular administration of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrospheres, the concentration of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein the systemic compartment was slightly above the EC₅₀ value (cellbased) during the first week, but then dropped below the EC₅₀ value forthe duration of the experiment (5-6 months). This experimentdemonstrates that3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrospheres provide sustained, local drug delivery to the knee jointwith low systemic (i.e. sub-therapeutic) drug exposure.

Example 4-2: Assessing the Pharmacokinetics of GZ389988 Following aSingle, Intra-Articular Injection of Three3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine—LoadedMicrospheres Extended Release Microsphere Formulations

Three formulations were selected to compare the in vitro and in vivopharmacokinetics in the systemic compartment following intra-articularinjection in rat knee joints. The formulations are presented in Table14.

In vitro release testing was performed using the method described inExample 3-1. In vivo performance was measured by injecting 3 male Wistarrats with a given microsphere formulation delivering either 0.1 or 1 mgof GZ389988 to the knee joint. At each time point, blood samples (˜0.25mL) were collected via a jugular vein cannula and placed into chilledtubes containing K₂EDTA as the anticoagulant, mixed, and kept on iceuntil centrifugation. The samples were centrifuged within 1 hour ofcollection at a temperature of 4° C., at 3,000×g for 5 to 10 minutes.Plasma was collected after centrifugation of the blood samples intopolypropylene tubes. Plasma samples were frozen on dry ice and storedfrozen at −60 to −80° C. prior to LC-MS/MS analysis. The concentrationof GZ389988 in rat plasma (expressed as ng/mL) was plotted over time (28days total).

TABLE 14 Compositions and Dose of Microspheres Tested in AssessingPharmacokinetics of GZ389988 Dose GZ389988† Administered FormulationBatch# Loading Polymer Ratio (mg) A 45 16% 9:1 0.1 R202H:RG752H A 46 16%9:1 1 R202H:RG752H B 47 15% 9:1 0.1 R203H:RG752H 48 15% 9:1 1R203H:RG752H C 49 40% R203H 0.1 50 40% R203H 1 †GZ389988 =3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine

The IVR profiles for Formulation and Formulation B show slow releasewith approximately 10% of the active compound released over 28 days.Formulation C shows slightly higher initial release over the first 5days, followed by a decreased release rate over the remaining 23 days;approximately 22% of the active compound was released over 28 days.

Following intra-articular administration into rat knee joints,Formulations A and B showed similar plasma-drug exposure profiles over28 days; both formulations showed T_(max) values of 1-1.5 hours with asteady-state plasma levels over the duration of the experiment (0.08 and0.8 ng/mL for the 0.1 and 1.0 mg doses, respectively). Compared withFormulations A and B, Formulation C showed a higher C_(max) value andhigher steady-state plasma level over the duration of the experiment(1.2 to 12 ng/mL for the 0.1 and 1.0 mg doses, respectively) (FIG. 29).These results were in good agreement with the IVR experiment results.

Example 4-3: Clinical Study to Assess the Safety, Tolerability, andPharmacokinetics of3-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein Patients with Painful Osteoarthritis of the Knee

The safety and efficacy of the monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein humans is tested in a two part double blind placebo-controlledclinical study to assess the safety, tolerability, and pharmacokineticsof single escalating intra-articular doses followed by assessment ofefficacy, safety, tolerability and pharmacokinetics of a singleintra-articular dose in patients with painful osteoarthritis of theknee.

In part one of the study, single intra-articular injections in the kneeof various doses of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminemonohydrate microcrystalline suspension, produced according to Example 2above, are tested against placebo doses of 3 mL and 5 mL.

Dose 3 mg/3 mL 10 mg/3 mL  30 mg/3 mL  60 mg/3 mL  100 mg/5 mL 

Adult men and women with a diagnosis of primary knee osteoarthritis areeligible for participation in the study. Patients are symptomatic formore than 6 months and provide written informed consent prior to anyprocedure related to the study. Efficacy is evaluated based on safetyand tolerability (adverse events, physical examination, body weight,body temperature, clinical laboratory tests, blood pressure, heart rate,12-lead electrocardiogram, local tolerance) at 12 weeks post-injection.Pharmacokinetics (plasma and, if possible, in synovial fluid) andpharmacodynamics are also evaluated.

Patients are followed for 84±7 days following study drug or placeboadministration, with option of continuing on in a long termobservational safety study with no additional study drug administrationto assess long term safety and efficacy.

Example 5: Polymorphism Study of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine

A polymorphism study of GZ389988 was conducted in order to identifycrystal forms of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein pure solvents and controlled mixtures of solvent and water. Thetechniques used as characterization tools of the polymorphs includedX-Ray Powder Diffraction (XRPD), High-Resolution X-Ray PowderDiffraction (HR-XRPD) and Single Crystal X-Ray Diffraction (XRSCD),Thermogravimetric Analysis (TGA) coupled with Infrared Spectroscopy(FT-IR) or Mass Spectrometry (MS), Dynamic Vapor Sorption (DVS) of waterand solvents, and Optical Microscopy (OM). These techniques aredescribed in greater detail below.

High Resolution X-Ray Powder Diffraction (Hr-XRPD)

High-resolution diagrams were recorded at ambient conditions on aPanalytical X'Pert Pro MPD powder diffractometer using theBragg-Brentano (vertical θ-2θ configuration) parafocusing geometrycoupled with a X'Celerator detector. A sealed copper anode X-ray tubewas used, running at 45 kV and 40 mA levels. An incident beammonochromator (Johansson type: a symmetrically cut curved germanium(111) crystal) produced pure Cu K α 1 radiation (λ=1.54060 Å).

For each set of experiments, a thin layer of the product was depositedonto the surface of a sample holder, covered with a single-crystalsilicon wafer. The wafer had been cut out according to Si (510)crystallographic orientation that, by systematic extinction, impeded anyBragg reflection from the silicon. The available angular range extendedfrom 2 to 50° in 2θ, with a 0.017° step size in 2θ. A variable countingtime from 100 to 2500 seconds per step was used.

X-Ray Powder Diffraction (XRPD)

Other XRPD analyses were carried out on a Siemens-Brucker D8 Advancepowder diffractometer, also using the Bragg-Brentano (vertical θ-θconfiguration) parafocusing geometry, and an Anton-Paar TTK450temperature chamber. A thin layer of the product was deposited onto asingle-crystalline silicon wafer, cut out according to Si(510)crystallographic orientation that, by systematic extinction, impeded anyBragg reflection from the wafer. A sealed copper anode X-ray tuberunning at 40 kV and 35 mA levels was used. Two lines were typicallyemitted: CuKα1 (λ=1.5405 Å) and CuKα2 (λ=1.5443 Å). A Nickel (3-filter,placed between the detector and specimen, did not altogether eliminateCuKβ (λ=1.3922 Å) radiation, which still contributed about 1% of thediffracted beam at the detector (manufacturer's data). The beam was sentthrough Soller slits to improve its parallelism. Variable divergenceslits kept the illumination of the sample area constant. A collimatorlimited the diffusion between the tube and the sample. A LynxEye lineardetector completed the setup. It had a 3.5°-wide detection window inangle 2θ. Diagrams were recorded in the following conditions: at ambienttemperature, scans from 2 to 40° in angle 2θ. Integration times dependedon experimental conditions. Evolution studies and most scans wereconducted using a 0.1 s second counting time per step in 2θ. Longerintegration times (up to 5 s) may have been used to characterize stableforms.

X-Ray Single Crystal Diffraction (XRSCD, Also Called SCXRD)

XRSCD data were recorded on a Bruker Smart Apex single crystaldiffractometer. A molybdenum IμS microfocus X-ray source was used,running at 50 kV and 0.6 mA, emitting Mo-Kα radiation (λ=0.710731 Å). ACharge-Coupled Device (CCD chip: 4K, 62 mm) area detector was positionedat 6.0 cm. An Oxford Cryosystems nitrogen cryostat (Cryostream Plus)allowed XRSCD experiments to be carried out at 100 K.

The crystals were both mounted from a Paratone N™ oil drop onto a lowbackground mylar MiTeGen loop. A full Ewald sphere of reflections wascollected (3 omega scans of 680 frames with a frame width of 0.3°).Accumulation times depended on the crystal.

The orientation matrix and unit cell were established using the Apex2(v2014.11-0) program suite. The 3D reflection profile and theintegration of all reflections were carried out with the SAINT (v8.34A)program. The SADABS (v2014/5) program was used to correct for Lorentzand polarization effects and for absorption by the sample. The tentativespace group was determined with the XPREP (v2014/2) program. The SHELXTL(v2014/7) suite was used to solve the structure by the intrinsic phasingmethod and to refine the solution by full-matrix least-squarescalculations on F².

Polymorph Identification and Characterization

Using the above described techniques, crystal forms of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminewere identified. These forms, XRPD 2θ peaks measured using CuK_(α)radiation and the conditions of formation are set forth in Table 15below.

TABLE 15 Forms of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine Anhydrous, Hydrate orPolymorph Form XRPD 2θ Peaks Measured Solvate Number Number usingCuK_(α) Radiation Conditions of Formation Anhydrous 1 Form 2  3.8, 7.6,11.0, 11.5, 13.3, Form obtained by 13.9 and 15.3 dehydration ofmonohydrate 1 Anhydrous 1 Form 3  6.0, 6.9, 10.9, 12.4, 12.7, Obtainedat high temperature 13.7 and 15.3 (160° C. for monohydrate 1, ~120° C.for solvates) Anhydrous 3 Form 6/7 5.6, 8.1, 12.1, 14.0, 16.2 and Formobtained by 20.7 desolvation of monohydrate 2 and of ethanol,acetonitrile and acetone 2 solvates Monohydrate 1 Form 1  3.6, 7.1, 8.9,10.4, 10.7, See Example 1 12.4, 12.7 and 14.3 Monohydrate 2 Form 8  5.5,6.4, 8.2, 12.5, 12.8 and Form deriving from ethanol, 16.5 acetonitrileand acetone 2 solvates (from “N₂/H₂O vapor” cycles) Sesquihydrate Form4  3.5, 7.1, 9.3, 10.3, 10.6, Form obtained by 12.4, 13.0 and 14.1crystallization from acetone/water Ethanol Form 5  5.3, 6.3, 7.5, 11.4,12.6, 12.8 Form obtained by Solvate and 15.1 recrystallization ofmonohydrate 1 in ethanol or from ethanol solvation of anhydrous form 3Acetone 1 Form 9  6.1, 8.9, 12.1, 15.3, 16.4, Form obtained by Solvate18.2 and 21.2 recrystallization of monohydrate 1 in pure acetone andacetone/water 99:1 Acetone 2 Form 10 5.4, 6.2, 7.4, 11.4, 12.6 and Formobtained by Solvate 14.8 recrystallization of monohydrate 1 in pureacetone or from acetone solvation of anhydrous form 3 Acetonitrile Form11 5.3, 6.4, 7.7, 11.6, 12.8, 13.2 Form obtained by Solvate and 15.5recrystallization of monohydrate 1 in acetonitrile or from acetonitrilesolvation of anhydrous form 3

Measured HR-XRPD diagrams of monohydrate Form 1, anhydrous Forms 2 and3, as well as a simulated XRPD diagram of sesquihydrate Form 4, areplotted together in FIG. 30.

A suspension of 20 mg/ml of GZ389988A form monohydrate in mix solventethanol/water (99/1) was heated up at 80° C. for 1 h. The suspension wasthen filtered. Next, the solution was kept overnight at roomtemperature. After a night, crystals were observed, later identified ascorresponding to the ethanol solvate form (labelled as Form 5). Thephysical quality of the crystal obtained by slow evaporation of a mix ofethanol and water (99/1) was appropriate to be analyzed by singlecrystal X-ray diffraction. The measurement was performed at 100K on a5×50×200 μm³ crystal, with an accumulation time of 300 s per frame.Diffractograms confirmed the crystallinity of the analyzed particle.FIG. 31 shows the HR-XRPD diagram of ethanol solvate powder (bottom)measured at room temperature, compared with XRPD diagram simulated fromXRSCD data measured at 100K (top).

A suspension of 20 mg/ml of GZ389988A form monohydrate 1 (Form 1) wasprepared in a mix of solvents acetone/water (99/1) or in pure acetone,and heated up at 80° C. for 1 h to achieve full dissolution. Next, thesuspension was filtered. The solution was then kept at room temperatureor at 5° C. After one night, crystals were visible to the bare eye,later identified as corresponding to an acetone solvate form (labelledas Form 9). The physical quality of the crystal obtained by slowevaporation of a mix of acetone and water (99/1) was appropriate to beanalyzed by single crystal XR diffraction. The measurement was performedat 100K on a 100×200×2000 μm₃ crystal, with an accumulation time of 30 sper frame. FIG. 32 displays the XRPD diagram for acetone solvate powder,measured at room temperature (bottom) and simulated from XRSCD at 100K(top).

In another experiment, a suspension of 20 mg/ml of GZ389988A formmonohydrate 1 (Form 1) was prepared in pure acetone, and heated up at80° C. for 1 h. Next, the suspension was filtered, and then directlycooled down to 5° C. and stored at that temperature. After one night,crystals were visible to the bare eye and similar to those obtained foracetone solvate 1, Form 9. After grinding them in suspension in amortar, they were identified as corresponding to another acetonesolvate, Form 10. FIG. 33 compares the XRPD for the two acetone solvateforms, Form 9 (bottom) and Form 10 (top).

A suspension of 20 mg/ml of GZ389988A form monohydrate in acetonitrile(ACN) was heated up at 80° C. for 4 h. The suspension was then filtered.Next, the solution was kept overnight at 40° C., then left for 2 hoursto cool down to room temperature. After a night, clusters of crystalshad formed at the bottom of the vial. The HR-XRPD diagram of theacetonitrile (ACN) solvate form (Form 11) is reported in FIG. 34.

Identification of Solid Crystalline Phase Forming in Acetone/Water andAcetonitrile/Water Mixed Solvents

50 mg of GZ389988A Monohydrate 1 were complemented with 2 mL of amixture of a solvent (acetone or acetonitrile) and demineralized water,at three different weight ratios: 50/50, 80/20 and 95/5. With acetone,additional mixtures at ratios 99/1 and 98/2 were probed. After 2 hoursat 80° C., the samples were filtered on a PTFE syringe filter with anominal pore size of 0.45 μm, and stored again at 80° C. for 15 minutesafter filtration.

Samples were then left to cool down overnight at 40° C., and then atroom temperature for another 24 hours.

Samples were then analyzed by XRPD in a chamber saturated with thecorresponding solvent. If necessary, large crystals were crushed in thevial with a spatula into a finer powder. A sample of “wet” powder wasthen deposited as flat as possible on the sample holder. The results ofthese analyses are presented in Table 16 below.

TABLE 16 Solid crystalline Phase of GZ389988A in Acetone/Water andAcetonitrile/Water Mixtures Solvent Solvent/Water weight ratioCrystalline phase(s) Acetone 99/1 Acetone solvate Form 9 98/2Monohydrate 1 97/3 Monohydrate 1 95/5 Monohydrate 1  80/20 Monohydrate 1 50/50 Monohydrate 1 Acetonitrile 95/5 Monohydrate 1  80/20 Monohydrate1  50/50 Monohydrate 1

Formation of solvates and hydrates from crystallization of Monohydrate 1in ethanol, acetone, acetonitrile and acetone/water andacetonitrile/water mixtures was studied and presented in this example.Ten crystalline phases have been identified:

-   -   Anhydrous Phase 1 (Form 2)    -   Anhydrous Phase 2 (Form 3)    -   Anhydrous Phase 3 (Form 6/7)    -   Monohydrate 1 (Form 1)    -   Monohydrate 2 (Form 8)    -   Sesquihydrate (Form 4)    -   Ethanol solvate (Form 5)    -   Acetone solvate 1 (Form 9)    -   Acetone solvate 2 (Form 10)    -   Acetonitrile solvate (Form 11)    -   The corresponding diffractograms are plotted together in FIG.        35.

From the polymorphism study conducted on ethanol, acetone andacetonitrile, as well as on solvent mixes (acetone/water andacetonitrile/water), several conclusions can be drawn. All three puresolvents lead to the formation of solid crystalline solvate phases.Recrystallization of GZ389988A in ethanol and acetonitrile each lead toone solvate form. Two solvate forms have been obtained fromrecrystallization in acetone. Crystals formed in acetone/water mixedsolvent systems with weight ratios of 98:2, 97:3 95:5, 80:20 and 50:50are all in the monohydrate 1 crystalline phase. Crystals formed inacetonitrile/water mixed solvent systems with weight ratios of 95:5,80:20 and 50:50 are also all monohydrate 1 crystals. Acetone solvate 1has been observed to transform into a mostly amorphous solid upondesolvation. If molecular mobility is increased by the presence ofvapors of mixed acetone and water, both the initial acetone solvate 1crystals and the amorphous solid reorganize into monohydrate 1. Ethanol,acetonitrile and acetone solvate 1 exposed to a temperature of 120° C.under nitrogen desolvate into the same anhydrous crystalline phase 2.Isomorphism is observed for ethanol, acetonitrile and acetone solvate 2forms (to be confirmed by single crystal X-ray diffraction). They allreversibly desolvate into the same anhydrous phase 3. Anhydrous phase 3hydrates into a monohydrate form, “monohydrate 2”, different frommonohydrate 1.

The invention claimed is:
 1. A method of manufacturing a crystallineform of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminecomprising: a. Mixing3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine with a solvent to form asuspension; b. Stirring the suspension; c. Collecting the solids in thesuspension by filtration; and d. Drying the solids.
 2. The methodaccording to claim 1, wherein the solvent comprises a mixture of acetoneand water.
 3. The method according to claim 1, wherein the suspension isstirred overnight.
 4. The method according to claim 1, wherein thesolids are air dried.
 5. The method according to claim 1, wherein thesolvent comprises a mixture of acetone and water, the suspension isstirred overnight, and the solids are air dried.
 6. A method ofmanufacturing a monohydrate form of3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminecomprising: a. Mixing3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine with a solvent to form asuspension; b. Stirring the suspension; c. Collecting the solids in thesuspension by filtration; and d. Drying the solids.
 7. The methodaccording to claim 6, wherein the solvent is a mixture of acetone andwater.
 8. The method according to claim 6, wherein the suspension isstirred overnight.
 9. The method according to claim 6, wherein thesolids are air dried.
 10. The method according to claim 6, wherein thesolvent is a mixture of acetone and water, the suspension is stirredovernight, and the solids are air dried.
 11. A method of manufacturing3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrocapsules by solvent extraction, comprising: a. Dissolving the3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein an organic solvent to form a drug solution; b. Adding a polymer tothe drug solution to form apolymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminesolution; c. Mixing thepolymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminesolution into an aqueous solution to form an emulsion; d. Addingdeionized water to the emulsion; e. Forming microspheres from theemulsion by solvent extraction; and f. Sieving the resultingmicrospheres using a surfactant solution.
 12. The method according toclaim 11, wherein the organic solvent comprises (i) dichloromethane andmethanol, (ii) dichloromethane, (iii) benzyl alcohol and methanol, (iv)dichloromethane and benzyl alcohol, (v) chloroform, (vi) chloroform andmethanol, or (vii) chloroform and benzyl alcohol.
 13. The methodaccording to claim 12, wherein the organic solvent comprisesdichloromethane and methanol.
 14. The method according to claim 12,wherein the organic solvent comprises dichloromethane, benzyl alcoholand methanol.
 15. The method according to claim 12, wherein the organicsolvent comprises dichloromethane and benzyl alcohol.
 16. The methodaccording to claim 11, wherein the polymer comprises poly(D,L-lactide).17. The method according to claim 11, wherein the polymer comprisespoly(D,L-lactide-co-glycolide).
 18. The method according to claim 11,wherein the polymer comprises poly(D,L-lactide) andpoly(D,L-lactide-co-glycolide).
 19. The method according to claim 11,wherein the aqueous solution comprises polyvinyl alcohol in water. 20.The method according to claim 11, wherein the surfactant solutioncomprises poloxamer 407 in water, polysorbate 80 in water, orpolysorbate 20 in water.
 21. The method according to claim 20, whereinthe surfactant solution comprises poloxamer 407 in water.
 22. A methodof manufacturing3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-amine-loadedmicrocapsules by solvent extraction, comprising: a. Dispersing the3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminein an organic solvent to form a drug suspension; b. Adding a polymer tothe drug suspension to form apolymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminedispersion; c. Mixing thepolymer/3-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)-6-(1-methyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-aminedispersion with an aqueous solution to form an emulsion; d. Addingdeionized water to the emulsion; e. Forming microspheres from theemulsion by solvent extraction; and f. Sieving the resultingmicrospheres using a surfactant solution.
 23. The method according toclaim 22, wherein the organic solvent comprises (i) ethyl acetate, (ii)dichloromethane, (iii) chloroform, (iv) ethyl acetate anddichloromethane, (v) ethyl acetate and chloroform, (vi) dichloromethaneand chloroform or (vii) ethyl acetate, dichloromethane and chloroform.24. The method according to claim 23, wherein the organic solventcomprises ethyl acetate.
 25. The method according to claim 23, whereinthe organic solvent comprises dichloromethane.
 26. The method accordingto claim 23, wherein the organic solvent comprises dichloromethane andethyl acetate.
 27. The method according to claim 22, wherein the polymercomprises poly(D,L-lactide).
 28. The method according to claim 22,wherein the polymer comprises poly(D,L-lactide-co-glycolide).
 29. Themethod according to claim 22, wherein the polymer comprisespoly(D,L-lactide) and poly(D,L-lactide-co-glycolide).
 30. The methodaccording to claim 22, wherein the aqueous solution comprises polyvinylalcohol in water.
 31. The method according to claim 22, wherein thesurfactant solution comprises poloxamer 407 in water, polysorbate 80 inwater, or polysorbate 20 in water.
 32. The method according to claim 31,wherein the surfactant solution comprises poloxamer 407 in water.